Antibodies for identifying and/or isolating at least one cell population

ABSTRACT

Monoclonal antibodies, or fragments thereof, are used for isolating and/or identifying at least one cell population. The cell population can include any of the following types of cells: haematopoietic stem cells, neuronal stem cells, neuronal progenitor cells, mesenchymal stem cells and mesenchymal progenitor cells. The antibodies, or fragments thereof, bind to an antigen which is the same as that bound by an antibody which is produced by the hybridoma cell lines CUB1, CUB2, CUB3 and CUB4, which were deposited in the DSMZ under the numbers DSM ACC2569, DSM ACC2566 and DSM ACC2565, on Aug. 14, 2002, and DSM ACC2551, on Jul. 12, 2002.

FIELD OF THE INVENTION

The present invention relates to a monoclonal antibody, or a fragmentthereof, for isolating and/or identifying at least one cell populationwhich is selected from the group comprising haematopoietic stem cells,neuronal stem cells, neuronal pre-cursor cells, mesenchymal stem cellsand mesenchymal precursor cells.

DESCRIPTION OF THE RELATED ART

The term “stem cell” denotes, in a general manner, any cell which hasnot yet differentiated and which possesses the ability both to produceidentical descendants and to differentiate into specific developmentallines.

Adult stem cells have the function of maintaining cell numberhomeostasis in the tissue concerned, i.e. of replacing cells which havedied. For this reason, stem cells are particularly to be found intissues which are subjected to high stresses. Adult stem cells have beenfound in a very wide variety of tissues and organs, such as, forexample, bone marrow, brain, liver, skin, intestine, cornea, etc.

In the bone marrow, haematopoietic stem cells produce new cellscontinuously since these latter cells are constantly required in theblood owing to the limited life span of most of the cells.

The starting point for the formation of blood cells is the pluripotent,undifferentiated haematopoietic stem cell which is still not determinedfor a specific function. When stem cells differentiate, precursor cells,which are unable to replicate themselves and only bring a specializedcell type to maturity, are formed first of all. Neither the pluripotentstem cell nor the different intermediate stages are able to fulfillcell-specific haematopoietic functions; it is only the cells which havematured which are able to do this. Progenitor cells which have enteredupon a particular differentiation route then also keep to this routeuntil maturation is achieved (commitment).

In addition to stem cells for haematopoietic cells, stem cell-like cellswhich are progenitors of nonhaematopoietic tissues are also present inthe bone marrow. These progenitors of non-haematopoietic tissues wereoriginally termed, inter alia, tissue culture plastic-adherent cells andhave more recently been termed either mesenchymal stem cells or bonemarrow stroma cells (MSCs).

These cells are of interest not only because of their multipotency asregards differentiation; they are also of interest, for example, fortheir possible use in cell therapy and gene therapy.

The fact that, under certain conditions, mesenchymal stem cells can alsodifferentiate into nerve cells means that, inter alia, there is a needto be able to distinguish these mesenchymal stem cells from neuronalprogenitor cells.

These neuronal progenitor cells (termed NPCs below) are found in thecentral nervous system. They also express Nestin and are able todifferentiate into neurones, astrocytes and oligodendrocytes.

Neuronal progenitor cells are CD133-positive; this cell surface markerwas originally found on haematopoietic stem cells. However, it hasrecently been shown that this marker is also expressed by nervous tissueand skeletal muscle tissue. For these reasons, this marker is notsuitable for distinguishing between different stem cells or progenitorcells on its own.

Since, as has been mentioned, haematopoietic stem cells continuouslygenerate new cells in the bone marrow, stem cells coexist with theprogenitor cells at the same time in the bone marrow. In the bonemarrow, these cells are present in a complex arrangement, thereby makingit difficult to identify rare cells. Stem cells and their directdescendants express a phenotype which is virtually identical. For thesereasons, it is not possible, either, to identify an ultimate stem cellsimply on the basis of visible features.

The frequency of stem cells in the bone marrow is from 1×10⁻⁵ to 1×10⁻⁶.In addition, the stem cells are as a rule widely scattered in the giventissue, which means that they are difficult to detect.

As has been mentioned above, haematopoietic stem cells divide, undercertain conditions, into progenitor cells whose further differentiationis to some degree already determined. Depending on the nature andquantity of the cytokines which are present, these myeloid and lymphoidprogenitor cells can in turn generate a variety of other progenitorcells which are, however, no longer able to replicate themselves.Examples of cytokines which regulate haematopoiesis are granulocytecolony-stimulating factor (G-CSF), macrophage colony-stimulating factor(M-CSF), several interleukins, stem cell factor (SCF), erythropoietin(EPO), etc.

In order to investigate the haematopoietic (blood cell-forming)potential of stem cells, relevant human cell populations aretransplanted into immunodeficient mice (NOD/SCID mice). If thetransplanted cells are stem cells, it is then possible to detect humanhaematopoiesis in addition to the murine haematopoiesis. This in-vivoassay is used to characterize and identify stem cells by in factanalysing the progeny of individual cells.

As can be seen from the above, haematopoietic stem cells possess greattherapeutic potential and are used in patients in whom the immune systemis impaired or completely destroyed.

FACS (fluorescence-activated cell sorter) can be used, for example, topurify haematopoietic stem cells from the bone marrow. This purificationdepends on the presence, on the stem cells, of particular cell surfaceproteins which distinguish the haematopoietic stem cells and theprogenitor cells from other cell types and on the absence of other cellsurface proteins, these latter proteins then being characteristic fordifferentiated haematopoietic cells. Each of the surface proteins bindsa different monoclonal antibody, with each of these antibodies beingconjugated to a different fluorescent dye, thereby making it possible touse FACS to separate the cells.

The cell surface marker CD34, in particular, has been used in the pastfor isolating haematopoietic stem cells.

In addition, antibodies directed against the antigen CD133 have recentlybeen used for characterizing haematopoietic stem cells. Miraglia et al.,“A novel five-transmembrane hematopoietic stem cell antigen: isolation,characterization and molecular cloning”, Blood 90: 5013-5021, (1997)have shown that this antigen is a 120 kDa glycoprotein which possessesfive transmembrane domains and which is expressed not only onhaematopoietic stem cells and their progenitors but also on neuronal andendothelial stem cells.

CD133 antibodies are used, in addition to the conventional CD34antibodies, for positively selecting haematopoietic stem cells andprogenitor cells on a clinical scale. CD133 is only expressed onCD34^(bright) (high fluorescence intensity) stem cells and progenitorcells. CD34^(bright) CD133-positive cells are in the main negative forother erythroid progenitor cell markers such as CD36 and glycophorin A.In addition to stem cells which induced human haematopoiesis in: theNOD/SCID mouse model, the majority of granulocyte/macrophage progenitorcells have also been found in CD133-positive fractions derived fromhuman bone marrow and peripheral blood.

SUMMARY OF THE INVENTION

In view of the above, it is an object of the present invention toprovide a novel monoclonal antibody which can be used to selectivelyisolate and/or characterize particular cell populations, in particularhaematopoietic stem cells and also neuronal and mesenchymal stem andprogenitor cells.

According to the invention, this object is achieved by means of amonoclonal antibody, or a fragment thereof, with the antibody, or thefragment thereof, binding to the same antigen as does an antibody whichis produced by the hybridoma cell lines as CUB1, CUB2, CUB3 and CUB4,which were deposited in the Deutsche Sammlung fur Mikroorganismen andZellkulturen [German collection of microorganisms and cell cultures](DSMZ), in accordance with the Budapest treaty, under the numbers DSMACC2569, DSM ACC2566 and DSM AC2565, on Aug. 14, 2002, and DSM ACC2551,on Jul. 12, 2002.

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from date of deposit. The deposit will be made available byDSMZ under the terms of the Budapest Treaty, and subject to an agreementbetween Applicant and DSMZ which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14). Availability of the deposited strains is not to beconstrued as a license to practice the invention in contravention of therights granted under the authority of any government in accordance withits patent laws.

The object underlying the invention is fully achieved in this way.

The inventors were able to demonstrate, in their own experiments, thatit is possible to use the novel antibodies according to the invention toisolate and characterize haematopoietic stem cells, mesenchymal stemcells and progenitor cells and neuronal stem cells and progenitor cells.In addition to this, the selectivity of the novel antibodies was foundto be similar to that of an antibody directed against CD133.

One object of the invention relates, in particular, to monoclonalantibodies, or fragments thereof, which are produced by the hybridomacell lines CUB 1, CUB2, CUB3 and CUB4.

The inventors were surprisingly able to isolate the antibodies using theantigen CDCP1.

CDCP1 is a plasma membrane protein which possesses three potential “CUB”domains. These domains are immunoglobulin-like domains which aredesignated by the initial letters of the first three moleculespossessing such domains which were identified. Proteins which possessthese domains are known to be preferentially expressed in the embryonicstage and in early developmental stages.

The CDCP1 gene encoding this protein (“CUB domain-containing protein”;SEQ ID NO: 1) has been described by Scherl-Mostageer et al.,“Identification of a novel gene, CDCP1, overexpressed in humancolorectal cancer”, Oncogene 20: 4402-4408, (2001). This research groupshowed that this protein, or its mRNA, is strongly over-expressed incancer types or tumours such as intestinal cancer and lung cancer. Onthe basis of its three-dimensional structure, it was identified as beinga transmembrane protein which possessed three CUB domains in theextracellular domain, and the authors proposed that it was involved, inparticular, in cell adhesion or interaction with the extracellularmatrix (see Scherl-Mostageer et al.).

However, the possibility that this protein might also be expressed, inparticular, on primitive haematopoietic stem cells in the bone marrowand peripheral blood and also on mesenchymal or neuronal stem cells andprogenitor cells is neither reported nor suggested in this publication.

Instead of the antibody which is in each case mentioned, it is accordingto a further object also possible, within the context of the presentinvention, to use a fragment of the antibody without this in each casebeing expressly mentioned. In this connection, fragment” is understoodas meaning any fragment of the antibody which retains theantigen-binding function of the antibody. Examples of such fragments areF_(ab), F_((ab′)2), F_(v) and other fragments such as CDR(“complementarity-determining region”, hypervariable region) fragments.The said fragments exhibit the binding specificity of the antibody andcan also be prepared recombinantly, for example using known methods.

The inventors were able to demonstrate that it is unexpectedly possibleto use the antibodies which are directed against the plasma membraneprotein CDCP1 to selectively characterize and isolate haematopoieticstem cells and mesenchymal and neuronal stem cells and progenitor cells.

When these novel antibodies were used to identify haematopoietic stemcells, it was found that the antibodies exhibited a selectivity whichwas superior to that of antibodies which are directed against CD34 andwhich is similar to that of the antibody which is directed againstCD133.

For this reason, the novel antibodies, or fragments thereof, provideadvantageous alternatives to the CD34 antibody when identifying orisolating haematopoietic stem cells.

Such monoclonal antibodies can be prepared using conventional methods(see Kohler and Milstein, “Continuous cultures of fused cells secretingantibody of predefined specifity”, Nature 256:495-497, (1975)).According to this method, an animal is immunized with an antigen, theantibody-producing cells are isolated from the animal and theseantibody-producing cells are fused with an immortal cell line. Theresulting hybridoma cell lines are screened to determine whether theyare able to produce an antibody against the antigen which was used forthe immunization.

According to an object of the invention, the antibodies according to theinvention also now make it possible to prepare further antibodies whichbind to the same antigen. Using the antibodies according to theinvention, it is possible to employ well known methods to isolate thecorresponding antigen structures and to develop further monoclonalantibodies against the same antigen structures, with the known methodsbeing employed in this case as well.

One object of the invention furthermore relates to hybridoma cell lineswhich are able to produce and release this type of antibody, inparticular the hybridoma cell lines CUB1, CUB2, CUB3 and CUB4.

In providing the novel antibodies, the inventors have, for the firsttime, made available monoclonal antibodies, as well as hybridoma celllines which produce and release these antibodies, which make it possibleto selectively detect cell populations which are expressing the CDCP1antigen. The antibodies therefore constitute a means, which is thus farunique and has many uses, for the physician and research worker todetect these types of cells, on the one hand, and, on the other hand, tomanipulate these cells, where appropriate, either using the antibodiesthemselves or using reagents which are coupled to them.

Another object of the invention furthermore relates to a method forisolating and/or identifying at least one cell population which isselected from the group consisting of haematopoietic stem cells,neuronal stem cells, neuronal progenitor cells, mesenchymal stem cellsand mesenchymal progenitor cells using an antibody, or a fragmentthereof, with the antibody, or the fragment thereof, binding to the sameantigen as does an antibody which is produced by the hybridoma celllines CUB 1, CUB2, CUB3 and CUB4, which were deposited in the DSMZ, inaccordance with the Budapest treaty, under the numbers DSM ACC2569, DSMACC2566 and DSM ACC2565, on Aug. 14, 2002, and DSM ACC2551, on Jul. 12,2002.

In another embodiment and according to yet another object, the methodaccording to the invention uses an antibody, or a fragment of anantibody, which is produced by the hybridoma cell lines CUB1, CUB2, CUB3and CUB4.

Another object of the invention furthermore relates to a method forisolating and/or identifying at least one cell population, which isselected from the group consisting of haematopoietic stem cells,neuronal stem cells, neuronal progenitor cells, mesenchymal stem cellsand mesenchymal progenitor cells, using an antibody, with the methodcomprising the following steps:

-   -   (a) bringing a sample of a cell suspension which contains at        least one cell population into contact with the novel monoclonal        antibody, or a fragment thereof, and    -   (b) isolating and/or identifying the cells which are linked to        the novel monoclonal antibody or to the fragment thereof.

Yet another object of the invention furthermore relates to a method forisolating and/or identifying at least one cell population, which isselected from the group consisting of haematopoietic stem cells,neuronal stem cells, neuronal progenitor cells, mesenchymal stem cellsand mesenchymal progenitor cells, using an antibody, with the methodcomprising the following steps:

-   -   (a) bringing a sample of cell suspension which contains at least        one cell population into contact with the novel monoclonal        antibody, or a fragment thereof, and with at least one        additional antibody which binds to at least one of the cell        populations, and    -   (b) isolating and/or identifying the cells which are linked to        the monoclonal antibody, or to the fragment thereof, and to the        additional antibody.

In this connection, the bringing into contact of a cell mixture with theantibody can according to a further object be effected in solution as isthe case, for example, when using a flow cytometer(=fluorescence-activated cell sorter (FACS)).

Described in a general manner, cells are loaded, in flow cytometry, withantibodies which are on the one hand specific for a surface marker andon the other hand coupled to a fluorescent dye. The cells which aremarker-positive fluoresce while the negative cells remain dark. It istherefore an object and possible to ascertain what proportion of a cellpopulation is marker-positive. At the same time, a flow cytometer makesit possible to record the size and granularity of cells.

It is also an object and possible to use a method for magnetic cellseparation (MACS, magnetic cell sorting). In this method, the cells arelabelled with magnetic beads, with it being possible for these beads tobe coupled to the antibodies, for example.

In addition, the bringing into contact can according to a further objectalso be carried out by immobilizing the monoclonal antibody on a supportas is the case, for example, in column chromatography.

The cell suspension can according to one object be any solutioncontaining bone marrow cells, blood cells or tissue cells.

After the cell suspension has been mixed with the antibody, the cellswhich are expressing the CDCP 1 antigen bind the antibody, after whichthese cells can, in contrast to the cells which have not bound anyantibody, be identified and/or isolated using the described methods.

In the method which was disclosed last, use is furthermore made of anadditional antibody which also recognizes the cells. This antibody canaccording to one object, for example, be an antibody which is directedagainst the CD90 marker, in the case of neuronal progenitor cells, andbe an anti-CD34 antibody, for example, in the case of haematopoieticcells. Using an additional antibody makes it possible toisolate/identify specific subpopulations, which consequently bind boththe novel antibody and additional antibodies, in particular antibodieswhich are already known. This method can according to one object beused, for example, to characterize the cells more precisely with regardto their surface markers.

The cell populations which have been isolated by the methods canaccording to a further object then be used to repopulate, by means oftransplantation, the bone marrow in immunosuppressed or immunodefectivepatients.

One object of the invention furthermore relates to the use of the novelantibodies, or fragments thereof, for isolating and/or identifying atleast one cell population which is selected from the group consisting ofhaematopoietic stem cells, neuronal stem cells, neuronal progenitorcells, mesenchymal stem cells and mesenchymal progenitor cells.

According to yet another object, particular preference is given to usingthe novel antibodies, or fragments thereof, in connection with analysingpatient samples, in particular tissue biopsies, bone marrow biopsiesand/or blood samples, and in particular, when classifying leukaemias.

In the present instance, it has been possible to use the novelantibodies to detect expression of the corresponding antigen onleukaemia blasts as, for example, in the case of acute lymphaticleukaemia (ALL), acute myeloid leukaemia (AML) and chronic myeloidleukaemia (CML).

According to another object, the invention furthermore relates to theuse of the CDCP-1 protein and/or of the nucleic acid which encodes theCDCP-1 protein for preparing antibodies, or fragments thereof, forisolating and/or identifying haematopoietic stem cells.

According to yet another object, the invention furthermore relates to apharmaceutical composition which comprises at least one novel antibody,or fragments thereof.

In addition to the antibody, which represents the active compound in thecomposition, this composition can according to another object alsocomprise suitable buffers, diluents or additives. Suitable buffersinclude, for example, Tris-HCl, glycine and phosphate, while suitablediluents include, for example, aqueous solutions of NaCl, lactose ormannitol. Suitable additives include, for example, detergents, solvents,antioxidants and preservatives. A review of the substances which can beused for compositions of this nature is given, for example, in: A.Kibbe, “Handbook of Pharmaceutical Excipients”, 3rd Ed., 2000, AmericanPharmaceutical Association and Pharmaceutical Press.

According to a further object, the invention furthermore relates to akit which comprises at least one novel antibody, or fragments thereof.

Further advantages ensue from the enclosed figures and the description.

It will be understood that the features which are mentioned above, andthose which are still to be explained below, can be used not only in thecombination which is in each case specified but also on their own orwith other combinations without departing from the scope of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments are depicted in the enclosed drawings and are explained inmore detail in the description, in which:

FIG. 1 a shows the reactivity of the antibodies according to theinvention with wild-type cells;

FIG. 1 b shows the reactivity of the antibodies according to theinvention with transfectants;

FIG. 2 a shows the coexpression of CD34 and CDCP1 on bone marrow cellpopulations (cells labelled with CD34-FITC) and CDCP1-PE (CUB 1));

FIG. 2 b shows bone marrow cells in a CD34 versus CD38 plot and gatingon the stem cell population (bone marrow cell populations labelled withCD38-FITC, CDCP1-PE, CD34-PerCP and CD133-APC);

FIG. 2 c shows the coexpression of CDCP1 and CD133 on CD34⁺/CD38⁻ bonemarrow stem cells;

FIG. 3 shows the expression of CDCP1 on mesenchymal stem cells;

FIG. 4 a shows the expression of CDCP1 on neuronal stem cells; and

FIG. 4 b shows the coexpression of CDCP1 and CD90 on neuronal stemcells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Preparing Transfectants

Starting with the cloning vector pBluescript II SK(+), into which thecoding region of human CDCP1 had been cloned (obtained from BoehringerIngelheim/Vienna), the coding sequence was subcloned into a pRK vector(PharMingen, San Diego, USA) and, at the same time, a five-foldmyc-epitope (13 amino acids of the c-myc protein) was attachedC-terminally. The pRK-CDCP1-myc5 DNA was cotransfected, together with apuromycin resistance plasmid (pSVpacAp: de la Luna et al., “Efficienttransformation of mammalian cells with constructs containing apuromycin-resistance marker”, Gene, 62(1): 121-126, 1988) into NIH-3T3mouse cells using the CaCl.sub.2 method.

After individual puromycin-resistant cells had been cloned, expressionof the CDCDP1-myc5 protein in the transfected cells was detected byWestern blotting using an anti-myc antiserum. The clone NIH-3T3/huCDCP1was subsequently selected.

Immunization

Two Balb/c mice were immunized four times intraperitoneally with approx.5-10×10⁶ NIH-3T3/huCDCP1 cells using a standard protocol. Three to fourdays after the last immunization, the spleen was removed and the spleencells were fused with SP2/0 myeloma cells using a standard protocol. Thecell culture supernatants from growing, HAT-resistant hybridoma cellswere tested by FACS analysis both on the transfectants and on thewild-type cell lines (NIH-3T3).

Supernatants which reacted selectively with the transfectants but notwith the wild-type cell line NIH-3T3 were judged to be specific forCDCP1. The corresponding hybridoma cells were cloned (limiting dilution)and positive clones were selected. Four clones (CUB1, CUB2, CUB3 andCUB4) which reacted selectively with NIH-3T3/huCDCP1 cells were obtained(isotypes: one IgG2b [CUB1] and three IgG2a [CUB2-4]).

The histograms in FIG. 1 a show that the four antibodies did not reactwith the wild-type cell. FIG. 1 b shows histograms in which thereactivity of the four antibodies CUB1, CUB2, CUB3 and CUB4 with thetransfectants is clearly depicted by the second, dark peak which ispresent in each case.

Investigating the Reactivity of the Antibodies on Peripheral Blood Cells

The reactivity of the antibodies on peripheral blood cells was testedfirst of all. The results of these investigations are shown in Table 1below.

It was found that CD3⁺ T lymphocytes, CD20⁺ B lymphocytes, CD56⁺ NKcells, CD14⁺ monocytes, neutrophilic granulocytes, Siglec-8⁺eosinophilic granulocytes, CD235a⁺ erythrocytes and CD61⁺ platelets werenegative for CDCP1.

TABLE 1 Reactivity of the CDCP1-specific antibody CUB1 on peripheralblood cells Cell Type Reactivity T lymphocytes (CD3⁺) − B lymphocytes(CD20⁺) − NK Cells (CD56⁺) − Monocytes (CD14⁺) − Neutrophilicgranulocytes (CD15⁺) − Eosinophilic granulocytes (Siglec-8⁺) −Basophilic granulocytes (CD203c⁺) − Erythrocytes (CD235a⁺) − Platelets(CD61⁺) −

The results of the reactivity of the antibodies on various cell lines,which are listed horizontally, are shown in Table 2 below. The fourantibodies tested, i.e. CUB1 to CUB4, are listed vertically.

Description of the Cell Lines:

K-562: erythroleukaemia; WERI-RB-1: retinoblastoma; BV-173: Pro-B cellleukaemia; HEL: erythroleukaemia; HL-60: promyelocytic leukaemia;KU-812: basophilic leukaemia; HepG2: hepatocellular carcinoma; MOLT-4:T-lymphocytic leukaemia.

TABLE 2 Reactivity of the CDCP1-specific antibodies CUB1, CUB2, CUB3 andCUB4 on various cell lines CD34- CD133- WERI- Tf* Tf* K-562 RB-1 BV-173HEL HL-60 KU-812 Hep-G2 MOLT-4 CUB1 − − + − − − − − − − CUB2 − − + − − −− − − − CUB3 − − + − − − − − − − CUB4 − − + − − − − − − − *Tf =Transfectant

It was found that all the cell lines tested were negative for CDCP1apart from K-562. Scherl-Mostageer et al. have already shown that K-562,an erythroleukaemic cell line, expresses CDCP1 mRNA.

Further investigations were carried out into the correlated expressionof CDCP1 and the other stem-cell markers CD34 and CD133 on varioussubsets of leukaemia blasts. The results obtained in theseinvestigations are summarized in Table 3 below. The expression patternswere obtained by means of standard immunofluorescence labellings, usingthe antibodies, and then carrying out FACS analysis.

The analysis showed that CDCP1 is an independent marker in relation tothe other stem cell markers since its expression is not necessarilycorrelated with that of the other markers. Frequent coexpression of allthree stem cell markers can be seen, in particular, in the case of themyeloid leukaemias (AML and CML).

TABLE 3 Expression of CDCP1 on leukaemia blasts ALL CML (B-ALL,Pro-B-ALL, AM (Blast Pre-B-ALL, C-ALL (M1-M5) crisis) CD34⁺DC133⁺CDCP1⁺2/20 4/11 4/10 CD34⁺DC133⁺CDCP1⁻ 5/20 2/11 0/10 CD34⁺DC133⁻CDCP1⁺ 1/201/11 2/10 CD34⁺DC133⁻CDCP1⁻ 7/20 0/11 2/10 CD34⁻DC133⁺CDCP1⁺ 0/20 2/110/10 CD34⁻DC133⁺CDCP1⁻ 0/20 0/11 0/10 CD34⁻DC133⁻CDCP1⁺ 1/20 0/11 1/10CD34⁻DC133⁻CDCP1⁻ 4/20 2/11 1/10

In the table, ALL denotes acute lymphatic leukaemia while AML denotesacute myeloid leukaemia and CML denotes chronic myeloid leukaemia (theadditional designations are classification and characterizationdesignations which are customarily used for the acute leukaemias).

The novel antibodies can consequently be used, for example, in routinediagnoses in connection with leukaemias.

Investigating the Reactivity of the Antibodies on Bone Marrow Cells

The reactivity of the antibodies with bone marrow cell populations wassubsequently investigated. It turned out that CDCP1 is exclusivelyexpressed on CD34⁺ stem cells and not on other populations (see FIG. 2a). The FACS-sorted CDCP1⁺ fraction consisted almost entirely ofimmature blasts and immature colonies: CFU-GM (colony-forming unitgranulocyte macrophage), BFU-E (burst-forming unit erythroid) CFU-GEMM(colony-forming unit granulocyte-erythroid-macrophage-megakaryocyte).

FIGS. 2 b and 2 c show the results of a four-colour analysis of bonemarrow cells.

In order to carry out the four-colour analysis, the cells were labelledwith the following antibody conjugates: CUB1-PE (phycoerythrin),CD133-APC (allophycocyanin), CD38-FITC (fluorescein isothiocyanate) andCD34-PerCP (peridin chlorophyll A protein).

In the plot in FIG. 2 b, CD34 is plotted against CD38. Stem cells arefound in the rare CD34⁺/CD38⁻ fraction (Terstappen and Huang “Analysisof bone marrow stem cell”, Blood Cells 20(1): 45-61, 1994). In the plotin FIG. 2 b, this population is shown in the “R2-region”.

In the plot in FIG. 2 c, CDCP1 is plotted against CD133. This plotdepicts the cells which can be seen in the “R2” region in the plot inFIG. 2 b. FIG. 2 c shows that essentially all the CD34⁺/CD38⁻ stem cellscoexpress CDCP1 and CD133.

In other experiments, the inventors were able to demonstrate that, 6weeks after CDCP1-positive cells had been transplanted into NOD/SCIDmice, human CD45⁺ cells had formed in the bone marrow of the mice.(CD45⁺ is a marker for haematopoietic cells). This consequently provesthat the cells which are isolated using the novel antibodies are able tocarry out haematopoiesis.

Investigating the Reactivity of the Antibodies on Neuronal andMesenchymal Stem Cells

In other experiments, the reactivity of the antibodies towards neuronaland mesenchymal stem cells was investigated.

Commercially available foetal neuronal progenitor cells (in thefollowing NPC) and mesenchymal stem cells, obtained from Cell-Systems,St. Katharinen, Germany, were used for this purpose.

In FIG. 3, the second dark peak in the histogram shows that the CUB2antibody reacts with mesenchymal stem cells. The histogram in FIG. 4 ashows the reactivity with NPC, with the antibody CUB2 in this case beinglabelled with PE (phycoerythrin).

In order to carry out the coexpression analysis, the cells were labelledwith the following antibody conjugates: CUB1+alqG2bPE (phycoerythrin),and CD90-APC (allophycocyanin). CD90 is known to be expressed on NPC(see, for example Vogel et al., “Heterogeneity among human bonemarrow-derived mesenchymal stem cells and neural progenitor cells”,Haematologica 88: 126-133, (2003)).

In the plot in FIG. 4 b, CDCP1 is plotted against CD90. It can be seenthat the majority of the NPC cells expressed CDCP1 (in addition to CD90as the “confirmation marker”).

In summary, therefore, these data show that CDCP1 is a novel marker forhaematopoietic stem cells and for mesenchymal or neuronal stemcells/progenitor cells. It is possible to use the antibody according tothe invention which is directed against this marker to selectCDCP1-expressing stem cells in a simple manner and then, for example,transplant them for the purpose of repopulating. The antibody accordingto the invention is consequently of very great importance for selectinghaematopoietic or mesenchymal and/or neuronal stem cells. In addition,it constitutes an outstanding alternative to the CD133 and CD34 markerswhich are commonly used for selecting stem cells.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of any appended claims. All figures, tables, and appendices, aswell as publications, patents, and patent applications, cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. An antibody that selectively binds to a proteinhaving the amino acid sequence of CUB domain-containing protein (CDCP1),or a fragment thereof.
 2. The antibody of claim 1, wherein the antibodyselectively binds to a protein having the amino acid sequence of SEQ IDNO: 1 or a fragment thereof.
 3. The antibody of claim 1, wherein theantibody is a monoclonal or a polyclonal antibody.
 4. The antibody ofclaim 2, wherein the protein having the amino acid sequence of SEQ IDNO: 1 is glycosylated.
 5. The antibody of claim 1, wherein the proteinhaving the amino acid sequence of CUB domain-containing protein (CDCP1)is not glycosylated or the fragment of the amino acid sequence of CUBdomain-containing protein (CDCP1) is not glycosylated.
 6. An isolatedprotein having the amino acid sequence of CUB domain-containing protein(CDCP1) that is glycosylated.